Intercellular adhesion molecule powder formulation

ABSTRACT

The present application discloses a finely divided, dry powdered pharmaceutical composition which is specially adapted to be administered as an insufflate which includes the following ingredients: 
     (a) an pharmacologically effective amount of sICAM-1; 
     (b) an amount of carboxymethyl cellulose which is effective to retain sICAM-1 on the intranasal membranes; 
     (c) an amount of a bulking agent which is effective to provide a bulking effect without exerting a significant effect on the retention of the sICAM-1 on the nasal passages.

RELATED APPLICATIONS

This application claims benefit of Provisional Application Ser. No.60/012,944, filed Mar. 6, 1996 and PCT International ApplicationPCT/US97/03,263, filed Mar. 4, 1997.

TECHNICAL FIELD

The present invention provides a novel composition for nasalinsufflation which provides a means for the administration of theextracellular portion of the Intercellular Adhesion Molecule (sICAM-1).

BACKGROUND ART

The sICAM-1 molecule and its antiviral effects are disclosed in EPA391088-A and U.S. Application Ser. No. 08/479,557, filed Jun. 7, 1995which are incorporated by reference. ICAM-1 is disclosed in U.S.5,284,931, which is incorporated by reference. This drug is active as acompetitive antagonist for rhinovirus binding. The intranasaladministration of sICAM-1 is mentioned in Ser. No. 07/514,033 but theconcept of formulating sICAM-1 as an insufflate for nasal administrationis not disclosed. Various diluents are mentioned for use in preparingcontrolled release pharmaceutical compositions of sICAM-1 includingcarboxymethylcellulose but there is no disclosure of the use of anyconcentration of carboxymethylcellulose sodium salt or of the concept offormulating a long acting nasal insufflate.

The normal nasal residence time for most drugs that are placed in thenasal passages is from 15 to 20 minutes. The residue of any drug that isnot systemically absorbed through the nasal membranes is cleared to theposterior portion of the nasopharynx and swallowed by the patient. Therelatively large molecular weight of sICAM-1 (82,000 daltons) preventsthe systemic absorption of any substantial amount of the drug from thenasal passages.

Insufflates are generally recognized as finely divided powders that aredirectly introduced into a body cavity by means of an insufflator orpowder blower to achieve a local or systemic effect. Insufflates mayalso be administered from a pressurized aerosol which provides accuratecontrol of the dose which is administered by using a metered aerosolvalve. In order to administer, by insufflation, a drug which is intendedto have a local effect in the nasopharynx, it is necessary to formulatethe drug in a solid diluent which is pharmaceutically acceptable anddoes not interfere with the dispersion of the drug, does not interferewith the adhesion of the drug to the mucus membranes, does not have anadverse effect on the shelf life of the drug and is not excessivelyhygroscopic so that it may cause the formation of large particles in thecomposition during storage under ambient conditions. For these reasonsit is desirable to provide a stable sICAM-1 therapeutic compositionwhich will enhance the retention of the protein in the nasal passages.

DISCLOSURE OF THE INVENTION

The applicant has discovered a novel composition for the intranasaladministration of sICAM-1 which is based on the use of a carrier systemwhich comprises carboxymethylcellulose sodium as a solid diluent for thesICAM-1.

Accordingly, it is a primary object of this invention to provide a drycomposition of sICAM-1 which is suitable for administration byinsufflation.

It is also an object of this invention to provide a dry composition ofsICAM-1 which does not interfere with the dispersion of the drug.

It is also an object of the invention to provide a dry composition ofsICAM-1 which promotes the adhesion of the drug to the mucus membranes.

It is also an object of the invention to provide a dry composition ofsICAM-1 which does not have an adverse effect on the shelf life of thedrug.

It is also an object of the invention to provide a dry composition ofsICAM-1 which is not excessively hygroscopic so that it may cause theformation of agglomerates in the composition during storage underambient conditions.

It is also an object of the invention to provide a dry composition ofsICAM-1 which is compatible with a hard gelatin capsule.

These and other objects of the invention will become apparent from areview of the appended specification.

MODES FOR CARRYING OUT THE INVENTION

The invention provides a finely divided, dry powdered pharmaceuticalcomposition adapted to be administered as an insufflate, said drypowdered pharmaceutical composition comprising:

(a) an therapeutically effective amount of sICAM-1;

(b) an amount of carboxymethyl cellulose which is effective to retainsICAM-1 on the intranasal membranes;

(c) an amount of a bulking agent to produce a pharmaceutically elegantproduct which is effective to prevent the collapse of thecarboxymethylcellulose and the sICAM-1 during lyophylization.

The formulation will be prepared to provide a delivered dose of from 0.1to 1.0 mg and preferably from 0.05 to 75 mg of sICAM-1 to the intranasalpassages by the introduction of one single inhalation of the insufflate.

sICAM-1 is a genetically engineered form of the extracellular portion ofthe sICAM-1 receptor and as such acts as a competitive antagonist forrhinovirus binding to host cells. This material is also known as BIRR 4and is a single chain recombinant, soluble glycoprotein derived bygenetic engineering from a naturally occurring transmembraneglycoprotein sICAM-1. BIRR 4 is a truncated form of the intercellularadhesion molecule 1(ICAM-1) containing the amino acids that comprise theextracellular domain of the molecule. Based on DNA, sequence analysis,BIRR 4 is predicted to contain five immunoglobulin-like domainsstabilized by intrachain disulfide bonds.

The premise in using sICAM-1 in the prevention and/or early treatment ofthe common cold is based on the fact that the sICAM-1 compound willprevent the rhinovirus from becoming attached to the mucous membrane ofthe nose. In order to protect the nasal membranes from the rhinovirus,it is desirable to substantially completely contact the nasal mucousmembranes with the sICAM-1 molecule in order that the sICAM-1 moleculewill be distributed across the mucous membrane surface where it willinteract with any rhinovirus particles that may be present in the nasalpassages and prevent or attenuate the clinical progress of anyinfection. The intended hosts for treatment with the product of theinvention include primates such as humans who may be treated to relievethe symptoms of a rhinovirus infection or for the prophylaxis of arhinovirus infection.

The applicants have discovered that the use of carboxymethylcellulosesodium as a carrier for sICAM-1 provides an insufflate which extends theaction of sICAM-1 in the nasal passages as compared to compositionswhich are based on aqueous solutions of sICAM-1 or which are insufflatesof sICAM-1 that are based on the use of hydroxypropyl cellulose orhydroxyethylcellulose. In addition, the applicant has discovered thatthe addition of a bulking agent which may be a sugar alcohol such assorbitol, mannitol or galactitol, or an amino acid such as glycine or apolymer such as a carbohydrate based polymer which may be dextran orcyclodextrin to the sICAM-1 combination with carboxymethylcellulosesodium will provide a bulking effect without exerting a significanteffect on the retention of the sICAM-1 on the nasal passages.

The particular carboxymethyl cellulose sodium used in the invention willbe a pharmaceutical grade that will have a minimum purity of 99.5%.Generally the preferred carboxymethyl cellulose sodium will have aviscosity in water of 150 to 250 cps when measured at a temperature of25° C. and a concentration of 1 wt %. If desired, carboxymethylcelluloses having different molecular weights may be blendedtogether to obtain a product having a desired viscosity.

The composition of the invention will also include inorganic salts whichwill provide a buffered pH of about 6.0. Generally, an alkali metalchloride such as sodium or potassium chloride is combined with sodiummonobasic phosphate and the pH is adjusted with an alkali metalhydroxide such as sodium hydroxide or potassium hydroxide.

The insufflate is preferably made by dissolving the components in waterand removing the water by lyophilization and passing the product oflyopilization through an appropriately sized sieve to provide a freeflowing powder which is finely divided and suitable for administrationusing a suitable inhaler.

The method in which the powder components are combined to form thelyophillizable composition is important because if the powders are notcombined according to the following procedure, the biological activityof the sICAM-1 may be adversely affected. It is preferred to prepare asolution of the sICAM-1 in the phosphate buffer by dialyzing the sICAM-1from the solution in which sICAM is produced into the phosphate buffersolution using a polysulfone diafiltration membrane. A solution of thecarboxy methyicellulose sodium and the bulking agent are separatelyprepared by dry blending the powdered components prior to dispersing themixed components in water at 45 to 55° C. and preferably at about 50° C.The dry blending of the bulking agent and the carboxymethyl cellulosesodium facilitates the wetting of the carboxymethyl cellulose sodium andminimizes the aggregation of the polymer when it is dispersed in water.After the powders are combined with water, it is preferred to employ ahigh shear mixing apparatus such as a Waring blender for 2 to 10 minutesto disperse the, powders in the water. The dispersed mixture is thenfiltered through an appropriate filter e.g 5 to 50 microns, and allowedto cool to room temperature.

After the polymer has cooled to room temperature, equal weights of thepolymer solution and the sICAM-1 solution are mixed and placed inlyophilization vials. The vials are loaded into a lyophilizer and freezedried.

Generally the compositions which are prepared for lyophilization willcontain:

Concentration wt % sICAM-1 0.1 to 1% Bulking agent 2 to 10%Carboxymethyl cellulose, Na 1 to 3% Alkali metal chloride 0.1 to 0.6.%Sodium Phosphate, monobasic 0.1 to 0.6% Sodium hydroxide qs. to pH 6.0Water qs* *sufficient to prepare aqueous solution (removed bylyophilization)

The dry powdered pharmaceutical composition will include in weight %:

sICAM-1 0.07 to 7% Bulking agent 70 to 90% Carboxymethyl cellulose, Na14 to 45%.

The preferred dry powdered pharmaceutical composition will include inweight %:

sICAM-1 1 to 5% Bulking agent 80 to 90% Carboxymethyl cellulose, Na 14to 21%.

Generally it will be preferred to package a single therapeutic dose ofthe powder in two piece hard gelatin capsules which may be opened justprior to use. Each capsule may contain from 5 to 20 mg of compositionwith from 0.01 to 1.0 mg of sICAM-1. The powder is placed directly in aninhaler or the intact capsule may be placed in an inhaler which isadapted to open the capsule and to expel the powder contents of thecapsule in a form where it is mixed with air and may be directlyinspired into the patient's nose.

For use in this type of an inhaler, the gelatin capsule should havehemispherical ends which interact with the inhaler to provide for properdispensing of the powder. An example of a useful apparatus for thepractice of the invention is described in U.S. Pat. No. 4,889,114, whichis incorporated by reference. That apparatus may be provided with anasal tip such as a DeVilbiss nasal tip, Model 40-TR,

The following example is added to illustrate a preferred embodiment ofthe invention and is not intended as a limiting description of theinvention.

EXAMPLE

The preparation of ICAM-1 is described in U.S. Pat. No. 5,284,931. Inorder to produce a truncated soluble derivative of ICAM-1 which lacksthe cytoplasmic domain, an in-frame stop codon (between the Dcyt and D5)is generated using oligonucleotide mutagenesis based on the method ofKunkel, Proc. Natl. Acad. Sci USA 82:488-492 (1985), as modified byPeterson et al. Cell 53:65-72 (1988) both of which are incorporated byreference. The oligonucleotide mutagenesis results in the formation of amutant ICAM-1 gene, designated Y452 E/F TAG, which, upon expressionresults in the formation of a truncated, secreted form of ICAM-1(sICAM-1).

An expression vector consisting of the hamster DHFR gene and the codingregion of the above described mutant ICAM-1 controlled by the promoter,splice signals and polyadenylation signal from the SV40 early region wasconstructed. The hamster DHFR gene was isolated for the plasmidpBR322DHFR (Mulligan, R. et al., Proc. Natl. Acad. Sci. USA78:2072-20766 (1981) by digestion with FspI and HindIII, followed byblunt end ligation into pSV2gpt (Mitchell et al, Mol. Cell. Biol.6:425-4490 (1986) cleaved with BamHI/HindIII. The mutant sICAM-1(soluble ICAM-1) cDNA was isolated by digestion with NotI. The ends werethen filled in using Klenow, and the molecules were then digested withHindIII. The molecules were then ligated with pBR322DHFR expressionvector (prepared by digestion with ApaI, ends then filled with Klenow,and digested with HindIII to remove the gpt gene). Thus, the sICAM-1gene was physically linked to the hamster gene in a SV40 expressionvector.

The completed vector was then transfected into Chinese hamster ovary(CHO) K1 DUX-B11 cells using the calcium phosphate coprecipitationmethod (Graham et al. Virology, 52:456-467 (1973). After two days ofgrowth in nonselective mediur, the cells were passaged in selectivemedium containing 0.05 to 2 μM of methotrexate, but lacking hypoxanthineand thymidine. Clones were isolated, subcloned, and tested for sICAM-1production by Elisa. Colonies secreting the greatest quantity of sICAM-1were then subjected to two further rounds of gene amplification, and astable cell line, designated CHO118A, was derived. This cell line whichis a preferred source of sICAM-1, secreted sICAM-1 into the culturesupernate to approximately 1 μg/ml.

The sICAM-1 was purified from supernates of CHO118A cells byimmunoaffinity chromatography with anti-ICAM-1 monoclonal antibody R6.5.For this purpose R6.5 was covalently coupled, to CNBr-activatedSepharose 4B (Pharmacia LkB) to a final concentration of 5 mg per ml ofpacked resin according to the manufacturers instructions. Allchromatographic steps were done at 4° C., and all buffers contained 0.2U/ml aprotinin and 1 mM of phenylmethylsulfonyl fluoride. One liter offiltered supernate containing approximately 1 mg of sICAM-1 was loadedinto a column of R6.5 Sepharose at a flow rate of 1 ml/minute. Thecolumn was then washed with 200 ml of 10 mM Tris/0.15 M NaCl at a flowrate of 2.5 ml/minute to remove unbound material. The bound sICAM-1 waseluted with 50 mM triethylamine/0.15 M NaCl/pH 11.0 at a flow rate of 1ml/minute. Fractions were collected and immediately neutralized by theaddition of 1M Tris, pH 6.0 to a final concentration of 20 mM.

Fractions containing the eluted sICAM-1 were identified by SDS-PAGE on10-15% polyacrylamide gradient gels followed by silver staining.Electrophoresis and staining were done using a Pharmacia Phastgel systemand silver staining kit according the manufacturers instructions. Thefractions containing sICAM were pooled and concentrated approximately 10fold using Centricon-30 microconcentrators (Amicon, Danvers, Mass.).

The protein content of one batch of purified sICAM-1 was determinedusing a Bio-Rad Protein Assay according to the manufacturer'sinstructions (Bio-Rad Laboratories, Richmond, Calif.) and this materialwas frozen in aliquots for use as reference standards. Subsequently, theconcentration of sICAM-1 in samples was determined in a “sandwich” typeELISA using those reference standards and two anti-ICAM-1 monoclonalantibodies, R6.5 and R6.1 (Rothlein et al. J. Immunol. 141:1665-1669(1988), that bind to non-overlapping epitopes. R6.1 was bound to theplastic in 96 well plates by incubating 10 μl of a 10 μg/ml solution for1 hour at 37° C. Each of the following steps was then done with 100 μlof reagent incubated at 37° C. for 20 minutes, followed by washes withphosphate buffered saline: (1) binding of serial dilutions of referencestandard sICAM-1 or unknowns; (2) binding of biotinylated R6.5 (1μg/ml); and (3) binding of horseradish peroxidase-conjugatedstreptavidin (Zymed Laboratories, South San Francisco, Calif.) at themanufacturers recommended concentration. After the addition of thesubstrate ABTS (Zymed), and incubation for 20 minutes at roomtemperature, the absorbance was determined at 410 nm in aspectrophotometer. The concentration was determined by comparison to areference standard curve.

sICAM-1 is dialyzed against a phosphate buffer using a polysulfonemembrane to provide a solution of sICAM-1 which contains a concentrationof 10 mg of sICAM-1; 3.4 mg sodium phosphate and 3.6 mg sodium chloride/1 ml of solution.

Carboxymethylcellulose (CMC) (20 g) and mannitol (100 g) are dry blendedtogether. Water (750 ml) preheated to 50° C. is agitated in a Waringblender set at low speed, and the dry blend of the CMC and the mannitolis added to the perimeter of the vortex in the Waring blender. As theviscosity increases, slowly increase the blender speed while continuingto add the powder blend. The mixture should be added over the course ofapproximately two minutes. When the powder addition is complete, thevolume of solution should be brought to one liter with the addition of50° C. water and mixing continued for 5 minutes. The warm mixture isfiltered through a 10 micron filter and allowed to cool to roomtemperature. Once the polymer solution is cooled to room temperature,equal weights of the carboxymethyl cellulose sodiummannitol solution aremixed with the sICAM-1 solution and 1 g of solution is placed in 5 mllyophilization vials. The vials are loaded into a lyophilizer and freezedried according to the following schedule:

step 1: product is placed on the lyophilizer shelf and the condenser isset to −50° C.;

step 2: when the condenser reaches −50° C., the vacuum is started andmaintained at 100 millitorr;

step 3: when the product temperature reaches −50° C., the shelftemperature is raised to −28° C. at a rate of 0.5° C./minute;

step 4: hold the temperature at −28° C. for 15 hours;

step 5: raise temperature to 25° C. at a rate of 0.5° C./minute;

step 6: hold temperature at 25° C. for 3.5 hours;

step 7: lower temperature to 4° C. at 0.5 °/minute

step 8: hold temperature at 4° C. until vials are stoppered undervacuum; and

step 9: remove product from lyophilizer.

The product from step 9 is sequentially passed through two US standardsieves (#18 and #35) to obtain a fine even powder. Thereafter 15 mg ofthe product is placed in a No. 3 two piece hard gelatin capsule.

The efficacy of the product was demonstrated in an animal model usingyoung farm pigs. To visualize the intranasal retention of the product,sICAM-1 was radiolabled with ¹²³I using a commercially availableiodination kit (Iodobeads, Pierce) and then incorporated into the CMCformulation and the control compositions listed below. Controlcompositions using a phosphate buffered saline solution having theformula: 50 mM monobasic phosphate, 104 mM sodium chloride, pH 6; ahydroxy propyl cellulose (HPC) power based composition having theformula: 1% wv HPC, 5% wv Mannitol; and a hydroxy ethyl cellulose (HEC)powder based composition have the formula: 1% wv HEC, 5% wv Mannitolwere prepared in an analogous manner as previously described for the CMCformulation. For the powder CMC and control compositions, thelyophilized materials were reduced in particle size to a fine powderusing a metal spatula and 30-50 mg of powder was filled into a 1 mlplastic disposable pipette tip. The pipette tip, attached to a hand heldpipette bulb, was inserted 1-1.5 cm into each nostril and the bulb wassqueezed to provide sufficient pressure to deliver the powders into theanimals nose. The phosphate buffered saline solution formulation wasadministered in a similar manner in 0.1 ml aliquots. All of theseclearance studies used approximately 20-50 microcuries of ¹²³I sICAM-1tracer. Following dosing, the animal was placed in front of an externalgamma scintigraphy camera and data was collected for 90 minutes. Rawcount data were corrected for decay and then converted to percentretention which was normalized to the initial deposited dose of ¹²³IsICAM-1. The results were as follows:

PERCENT OF DOSE RETAINED 90 COMPOSITION MINUTES AFTER ADMINISTRATIONSolution formulation 30% Carboxymethyl cellulose 80% sodiumHydroxypropyl cellulose 50% Hydroxyethyl cellulose 60%

These tests show that the retention time on the nasal membrane, for thecomposition of the invention, is surprising and unexpected.

The invention has been described in connection with the specificembodiments thereof and it will be understood that it may be modifiedand that this application is intended to cover any variations, uses oradaptations of the invention following, in general, the principles ofthe invention and including such departures from the present disclosureas come within known or customary practice within the art to which theinvention pertains and as may be applied to the essential featureshereinbefore set forth in the appended claims.

What is claimed is:
 1. A method of treating rhinovirus infection in a warm-blooded animal comprising using an insufflate for nasal administration and wherein the drug in such insufflate is soluble intercellular adhesion molecule, type I (sICAM-1), the improvement which comprises using as the insufflate a dry, powdered pharmaceutical composition comprising: (a) sICAM-1 is an amount of about 0.07 to about 7% by weight; (b) a bulking agent selected from the group consisting of amino acids, sugar alcohols and carbohydrate based polymers, in an amount of about 70% to about 90% by weight; and (c) carboxymethyl cellulose, Na, in an amount of about 14% to about 45% by weight, where the dry, powdered pharmaceutical composition is sequentially passed through U.S. standard sieves #18 and #35.
 2. The method as recited in claim 1 wherein the bulking agent in the dry, powdered pharmaceutical composition is a sugar alcohol selected from the group consisting of sorbitol, mannitol and galactitol.
 3. The method as recited in claim 1 wherein the dry, powdered pharmaceutical composition further comprises a phosphate buffer. 